The present invention relates generally to regulatory sequences and more specifically to a nucleic acid sequence for the CCR5 promoter.
Chemokines constitute a structurally-related family of secreted proteins, most of which chemoattract and activate specific subsets of leukocytes in vitro. Chemokines are classified into two major subfamilies depending on the position of the first two of four conserved cysteines, which are adjacent in the case of CC chemokines and separated by a single amino acid in the case of CXC chemokines. In vitro, CXC chemokines attract lymphocytes and neutrophils, whereas CC chemokines typically do not attract neutrophils, but instead attract monocytes, macrophages, eosinophils, basophils, dendritic cells and lymphocytes with variable selectivity and potency. In vivo, chemokines appear to act as locally-produced emergency signals that direct leukocytes to sites of infection and tissue injury, but they may also regulate basal leukocyte trafficking, hematopoiesis, angiogenesis and other processes. See, e.g., H. E. Broxmeyer et al., J Immunol (1993) 150:3448.
Chemokines activate leukocytes by binding to selective, seven-transmembrane-domain, G protein-coupled receptors present on the plasma membrane. To date, twelve functional human chemokine receptors have been identified: four are specific for CXC chemokines and eight are specific for CC chemokines. Most of the receptors identified so far bind multiple chemokines; conversely, most chemokines tested so far bind to two or more receptor subtypes. In addition to their suspected beneficial role in host defense and tissue repair, several chemokine receptors (CCR2B, CCR3, CCR5 and CXCR4) are exploited pathologically by HIV-1, acting together with CD4 as cell entry coreceptors in vitro (G. Alkhatib et al., Science (1996) 272:1955. The HIV-1 strain specificity of the coreceptors is complex. CCR5 is used preferentially by most primary isolates, and not by T cell line-adapted lab strains (Y. Feng et al., Science (1996) 272:872; T. Dragic et al., Nature (1996) 381:667; H. Deng et al., Nature (1996) 381:661; H. Choe et al., Cell (1996) 85:1135; B. J. Doranz et al., Cell (1996) 85:1149; C. C. Bleul et al., Nature (1996) 382:829; E. Oberlin et al., Nature (1996) 382:833; L. Zhang et al., Nature (1996) 383:768). CXCR4 is used preferentially by lab strains and by some primary isolates. CCR3 is used by both primary isolates and lab-adapted strains. Only a few strains are able to use CCR2B (Doranz et al., supra). The importance of this for HIV-1 transmission in vivo has been clarified for CCR5 by the discovery of a benign, inherited, non-functional mutant CCR5 allele that in homozygous individuals is associated with a high level of resistance to natural HIV-1 infection (M. Samson et al., Nature (1996) 382:722). Epidemiologic analysis of the CCR5 xcex9432 mutant indicates that it contains a 32 base pair (bp) deletion in the open reading frame (ORF), and encodes a non-functional protein (D. Carrington, et al., Science (1996) 273:1856; Y. Huang, et al., Nat Med (1996) 2:1240; R. Liu, et al., Cell (1996) 86:367; P. A. Zimmerman, et al., Mol Med (1997) 3:23). CCR5 xcex9432 is relatively common especially in Caucasians where the allele frequency is about 10%. Homozygotes are found in 1% of Caucasian random blood donors but at a much lower than expected frequency in HIV-1+ Caucasians (B. Wang, et al., Lancet (1997) 350:9079; M. W. Smith, et al., Lancet (1997) 350:9079; T. R. O""Brien, et al., Lancet (1997) 349:9060). Moreover, PBMCs from CCR5 D32 homozygotes that have been tested are not injectable in vitro with CCR5-tropic strains of HIV-1 (Liu, et al., supra). CCR5 xcex9432 heterozygotes may be partially protected against HIV-1 transmission by heterosexual intercourse, but may be minimally or not at all protected against perinatal transmission or transmission by homosexual intercourse (T. L. Hoffman, et al., J Infect. Dis. (1997) 176:1093; R. E. Edelstein, et al., J. Acquir. Immune. Defic. Syndr Hum. Retrovirol. (1997) 16:243; C. M. Rousseau, et al., J. Acquir. Immune. Defic. Syndr. Hum. Retrovirol. (1997) 16:239). In numerous studies of HIV-1 seroconvertors, progression to AIDS was delayed on average 2 years in CCR5 xcex9432 heterozygotes compared to individuals lacking this allele (N. L. Michael, et al., Nat. Med. (1997) 3:338). Further, specific agonists for CCR5 (MIP-1xcex1, MIP-1xcex2 and RANTES) are able to suppress infection of CCR5-expressing cells by appropriate HIV-1 strains (F. Cocchi et al., Science (1995) 270:1811). Thus, HIV-1+ individuals heterozygous for the mutant CCR5 allele appear to have slightly delayed progression to AIDS compared to individuals homozygous for the wild type allele.
Several other allelic variants of chemokine receptors and one for a chemokine ligand have also been described that affect HIV-1 pathogenesis. m303 is a rare variant of the CCR5 ORF (allele frequency less than 1%) that encodes a truncated receptor and that protects against HIV-1 infection when found in the compound heterozygous state with CCR5 xcex9432. 15,16 Other uncommon variants of the CCR5 ORF have been described but their significance is unknown (M. Carrington, et al., Am. J. Hum. Genet. (1 997) 61:126 1; M. A. Ansari-Lari, et al., Nat Genet (1997) 16:221).
An allelic variant of the HIV-1 coreceptor CCR2 (named CCR2-64I or 46295-G/A, allele frequencyxcx9c10%) is associated with delayed progression to AIDS in heterozygotes (L. G. Kostrikis, et al., Nat Med (1998) 4:350; M. W. Smith, et al., Science (1997) 277:959). This allele has a single base change that causes a conservative substitution in a transmembrane region. CCR2-64I has been linked to a base change in the CCR5 promoter (Kostrikis, et al., supra) however, the functional importance of this has not been defined, and the mechanism of action of CCR2-64I remains unknown. A variant allele of the chemokine SDF-1, which is a ligand for the HIV-1 coreceptor CXCR4, affects the rate of progression to AIDS in homozygous individuals (C. Winkler, et al., Science (1998) 279:389). The alteration in this allele is a single base change in the 3xe2x80x2-untranslated region of the SDF-1 mRNA.
CCR5 mRNA has been detected in peripheral blood mononuclear cells (PBMCs) and adherent monocytes. Using a specific monoclonal antibody, CCR5 protein has been detected in microglial cells of the central nervous system and memory T cells. CCR5 protein expression can be upregulated by treatment of T cells with IL-2. In contrast, CCR5 RNA and HIV-1 coreceptor function can be downregulated in CD4+ T cells by CD3/CD28 costimulation. Thus, measures designed to block CCR5 expression or function could be used to block HIV-1 transmission and/or to treat established HIV-1 infection. In this regard, detailed knowledge of the factors regulating CCR5 expression is an important goal.
The present invention is based on the identification of the structural organization and sequence of the CCR5 gene and a functional promoter capable of constitutive expression of CCR5 in both myeloid and lymphoid tissues.
One aspect of the invention is an isolated DNA molecule comprising the sequence of the CCR5 promoter and functional regions or portions thereof. Another aspect of the invention is a DNA molecule comprising the sequence of the CCR5 promoter, or a function portion thereof operably linked to a heterologous protein.
Another aspect of the invention is an oligonucleotide or oligonucleotide analog capable of binding to the CCR5 promoter and inhibiting the activity thereof
Another aspect of the invention is a method for inhibiting the activity of MIP-1xcex1, MIP-1xcex2, or RANTES by administering an effective amount of a CCR5 promoter-inhibiting oligonucleotide or oligonucleotide analog to a subject in need thereof. Another aspect of the invention is a method for treating or preventing HIV infection in a subject having or at risk of having HIV infection by administering an effective amount of a CCR5 promoter-inhibiting oligonucleotide or oligonucleotide analog to a subject in need thereof. Another aspect of the invention is a method for treating inflammation by administering an effective amount of a CCR5 promoter-inhibiting oligonucleotide or oligonucleotide analog to a subject in need thereof.
Another aspect of the invention is an oligonucleotide agent functional only in myeloid and lymphoid cells, comprising a CCR5 promoter operably linked to an active heterologous protein.
Another aspect of the invention is a method for ablating a selected myeloid or lymphoid cell, by administering an effective amount of an oligonucleotide agent comprising a CCR5 promoter operably linked to a gene encoding a cytotoxic agent.